1. Field of the Invention
The present invention relates to a process for the isolation and purification of caffeine-free catechins from a number of different biomass sources. More particularly, the present invention relates to a four-step process whereby highly pure, caffeine-free EGCG is isolated and purified in high yields from plant materials such as green tea leaves.
2. Description of the State of Art
Commercial green tea is made by steaming or drying fresh tea leaves at elevated temperatures. Its chemical composition is similar to that of fresh tea leaves. Green tea contains polyphenols, which include flavanols, flavandiols, flavonoids, and phenolic acids; these compounds may account for up to 30% of the dry weight. Most of the green tea polyphenols are flavanols, commonly known as catechins. Some major green tea catechins are (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), (-)-epicatechin (EC), (+)-gallocatechin, and (+)-catechin. The chemical structures of some of these compounds are shown in FIG. 1. Caffeine, theobromine and theophylline, the principal alkaloids, account for about 4% of the dry weight.
Many laboratory studies have demonstrated inhibitory effects of tea preparations and tea polyphenols against tumor formation and growth. This inhibitory activity is believed to be mainly due to the antioxidative and possible antiproliferative effects of polyphenolic compounds, and in particular the major catechins EGCG, EGC, ECG and EC in green tea. The major constituent and possibly the most powerful of these catechins is EGCG. These catechins may also inhibit ,carcinogenesis by blocking the endogenous formation of N-nitroso compounds, suppressing the activation of carcinogens, and trapping of genotoxic agents (Yang, C. and Wang, Z.-Y., J. National Cancer Institute 85:1038 (1993)).
Investigators have tested EGCG on cancerous human and mouse cells of the skin, lymph system, and prostate, and on normal human skin cells. In vitro, EGCG resulted in the induction of apoptosis, or programmed cell death, in cancer cells, but not in normal cells (Journal of the National Cancer Institute, 89, (1997)). In addition, it was noted that EGCG did not cause necrosis of healthy cells. This selectivity, if it can be observed in vivo at desirable doses, will be of great therapeutic importance, since a vast variety of chemotherapeutic agents currently used in cancer therapy are thought to kill cells by mechanisms other than apoptosis. Thus, green tea, and in particular its major component, EGCG, appear to be ideal agents for chemotherapy because they appear to have little or no known side effects.
Disadvantages to drinking green tea for health benefits includes the fact that individuals may need to consume large quantities of tea in order to receive even small therapeutic amounts of catechins. Further, brewed tea contains caffeine, which is known to have undesired effects on the cardiovascular system as well as a mutagenic effect.
Therefore, there is a need to isolate a caffeine-free composition of green tea catechins having a known amount of EGCG.
Antioxidants have been used in cosmetics, for example sun screens, to prevent or to lessen the amount of tissue damage due to free radicals. Free radical damage is often initiated by environmental factors such as exposure to UV light, which can increase the number of free radicals in the skin, which in turn can damage DNA. Free radical damage has been linked to the aging process in addition to chronic degenerative diseases including heart disease, arthritis, and cancer. Natural defense systems in biological systems react directly with the free radicals to prevent damage to tissue. Unfortunately, the body's antioxidant defense system is often not efficient enough to counter free radical production rates during periods of prolonged exposure to environmental factors. Vitamin E has been shown to assist in the repair of lipid peroxides formed by free radicals but does not prevent them from forming. Melatonin has been shown to protect against the effects of hydroxide radicals, but not oxygen radicals. Green tea catechins have been shown to not only prevent against lipid peroxidation but also scavenge both oxygen and hydroxide radicals.
In addition, antioxidants are widely used in pharmaceuticals, cosmetics, essential oils and plastics for food packaging. Butylated hydroxy anisole (BHA), a synthetic antioxidant, was formerly the most widely used antioxidant worldwide. However, because of its possible carcinogenicity, the use of BHA is banned or restricted in many countries. Tocopherol (Vitamin E) is an antioxidant from natural sources. Tocopherol is not carcinogenic, however, its lipophilic property limits its wider use. Therefore, there is a need for effective, safe, and natural antioxidants. Tea catechins are known to have antioxidative property. The antioxidative effects of four catechins have been tested in lard, and their antioxidative activity increased in the following order: EC&lt;ECG&lt;EGC&lt;EGCG.
Several methods have been described in the prior art for producing decaffeinated green tea or for isolating catechins from green tea. U.S. Pat. No. 5,043,100 (Chang, et al.) describes vacuum steam distillation of alcohol extracts of green tea. However, Chang et al. do not characterize the compositions of the distillates, nor do they indicate whether or not caffeine has been eliminated from the distillates.
U.S. Pat. No. 4,613,672 (Hara) describes a process for the production of a mixture of tea catechins comprising EC, EGC, ECG and EGCG by extracting tea leaves with hot water or an aqueous alcoholic solution, washing this extract with chloroform to remove caffeine, extracting catechins in the aqueous layer into an organic solvent and distilling the organic phase. This method, which uses a toxic solvent in the extraction process, does not provide for the isolation of EGCG in highly pure form from the mixture of catechins.
EP 547370(B1) (So) describes a method for preparing an antioxidant which involves extracting tea leaves with water and then fractionating the extract by means of liquid chromatography using water as the eluent. However, So does not indicate whether or not caffeine has been removed from the antioxidants and does not give the composition or purity of the antioxidant.
WO 96/28178 (Bombardelli, et al.) describes a method for the preparation of decaffeinated mixture of polyphenols by washing green tea extracts with chlorinated solvents. These mixtures comprise 50-65% EGCG, 13-20% ECG, 2-4% EC and 1.5-35 EGC, and this method does not provide for the isolation of EGCG in a highly pure form. In addition, this method uses a toxic solvent in the extraction process.
The above examples describe some of processes that currently exist for extracting and purifying catechins from various plant materials. However, in addition to some of the disadvantages already discussed, many of the disclosed processes are also not easily scaled up to an efficient commercial process where disposal considerations of various solvents play an important role in the overall feasibility of the process.
A further disadvantage of the processes disclosed in the prior art is the inability to achieve a high concentration and purity level of EGCG, and in particular, caffeine-free EGCG of high purity. Rather, the above processes result in mixtures of catechins having various concentrations of EGCG.
There is still a need, therefore, for an efficient process and procedure for isolating and purifying caffeine-free catechins of desired compositions for uses in pharmaceuticals, nutraceuticals and cosmetics which is cost-effective, scalable, and which does not require the use of toxic solvents. Further, there exists a need for a process for isolating and purifying a caffeine-free catechin mixture having a desired concentration of EGCG.